Dietary composition for preventing, reducing, alleviating or treating idiopathic vomiting

ABSTRACT

The present invention relates to compositions containing plant materials or extracts with inhibitory effect on a 5-HT 3a  and/or NK-1 receptor for preventing or treating idiopathic vomiting.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. application Ser. No.14/198,662, filed Mar. 6, 2014 (issued Oct. 15, 2019 as U.S. Pat. No.10,441,623), which claims benefit of U.S. provisional application Ser.No. 61/786,465, filed Mar. 15, 2013. This application is also acontinuation-in-part of U.S. application Ser. No. 14/198,669, filed Mar.6, 2014 (issued Feb. 4, 2020 as U.S. Pat. No. 10,548,935), which claimsbenefit of U.S. provisional application Ser. No. 61/786,485, filed Mar.15, 2013. Each of these applications is hereby incorporated by referencein its entirety herein.

FIELD

This invention is related to a dietary composition or a method forpreventing, reducing, alleviating, or treating idiopathic vomiting inmammals, particularly domestic cats, using one or more plant materialsor extracts that have inhibitory effect against the5-hydroxytryptamine-3 serotonin (5-HT3a) and/or the neurokinin-1 (NK-1)receptor.

BACKGROUND

Chronic/cyclic idiopathic emesis or vomiting syndrome in cats wasidentified in the late nineteenth century. Although infrequent vomitingby cats under certain circumstances may be acceptable, e.g., eating toofast or too much or presence of excessive hair or other foreign objectsin the stomach, frequent vomiting or regurgitation without causes(“idiopathic vomiting”) can result in severe malnutrition in cats andcause damages to the gastrointestinal (GI) health of the cats. The fourmain characteristics that define idiopathic vomiting are: 1) three ormore recurrent separated episodes of vomiting or regurgitation; 2)varying intervals of completely normal, healthy periods between theepisodes; 3) episodes are typical with regard to the timing of onset,symptoms and durations; and 4) unknown causes of vomiting orregurgitation. Subjects susceptible to idiopathic vomiting cannot beidentified by standard medical examination, including physicalexamination and/or blood work. In humans, idiopathic vomiting may bedescribed as Cyclic Vomiting Syndrome (CVS), and may be associated withdehydration, injury to the GI tract (particularly the esophagus), andtooth decay (vomitus may be acidic).

The exact causes of idiopathic vomiting are not fully understood.However, it has recently been recognized that one of the potentialcauses in cats is related to central nervous system (CNS) disorders.There are several neurotransmitter receptors in the brain of the catsthat can be triggered to stimulate or activate different biologicalpathways leading to emesis. Examples of such receptors includeneurokinin (NK) receptors, histamine receptors, acetylcholine receptors,serotonin receptors, mu-opioid receptors, and dopamine-2 receptors.Therefore, a potential way to prevent or reduce idiopathic vomiting isto inhibit or partially block such receptors.

Certain 5-HT_(3a) receptor antagonists, such as dolasetron, granisetron,ondansetron, and palonosetron, have demonstrated effectiveness as anantiemetics in humans and have been used to manage chemotherapy-inducednausea and vomiting in cancer patients. Further, a new class of drugsknown as NK-1 receptor antagonists has been recently developed forcontrolling emesis in humans, which include aprepitant and maropitant,among others. However, these compounds often lead to side effects.Further, it is difficult to administer such compounds through feeding,because of their undesirable taste. Unfortunately, injection is not aconvenient alternative means of administering antiemetics. When treatinganimals, such as cats or dogs, injection may require veterinaryassistance, particularly if the animal resists the injection.

Therefore, there is a continuing need for an effective and more readilyavailable treatment for preventing, reducing, alleviating, or treatingidiopathic vomiting. There is also a need for treatments with lesserside effects that can be easily administered such as, for example,through feeding or other oral administration. There is still further aneed for naturally-derived therapeutic compounds or compositions. Theseneeds are particularly acute for domestic cats with a history ofidiopathic vomiting.

SUMMARY

One aspect of the present invention meets the above-described needs byproviding a dietary composition for preventing, reducing, alleviating,or treating idiopathic vomiting in a companion animal, which containsone or more plant materials or extracts thereof in an effective amountfor inhibiting a 5-hydroxytryptamine-3a serotonin (5-HT_(3a)) receptorand/or the neurokinin-1 (NK-1) receptor.

The plant materials or extracts thereof consist of Curcuma longa and oneor more of licorice, ginger or rhubarb. The plant materials or extractsare provided in the dietary composition in amounts effective to resultin a 5-HT_(3a) and/or NK-1 receptor inhibition of from 30% to 55%. Thepet dietary composition is desirably a pet food and as such may furthercomprise a source of carbohydrate, a source of protein, and optionally asource of fat. In such a food, the one or more plant materials orextracts are present in amounts from 0.1 ppm to 75,000 ppm, or from 1ppm to 1000 ppm.

In some embodiments, the pet food may be in the form of dry kibbles, wetcanned food, gravies, or treats, while in others, the dietarycomposition may be provided in the form of a pet supplement. In suchembodiments, the pet supplement may comprise from 0.1% to 99%, or from0.5% to 15%, of the one or more plant materials or extracts, by weightof the supplement. The pet supplement may be in the form of a powder,liquid, syrup, pill, encapsulated composition, or combinations of these.

In another aspect, the present invention relates to a method forpreventing, reducing, alleviating, or treating idiopathic vomiting in acompanion animal, which includes the step of orally administering tosaid companion animal one or more plant materials or extracts thereof inan effective amount for inhibiting the 5-HT_(3a) receptor and/or theNK-1 receptor.

In a further aspect, the present invention relates to a composition forpreventing, reducing, alleviating, or treating idiopathic vomiting in acompanion animal, which contains one or more plant materials or extractsthereof in an effective amount for inhibiting the 5-HT_(3a) receptorand/or the NK-1 receptor.

In a still further aspect, the present invention relates to a packagefor preventing, reducing, alleviating, or treating idiopathic vomitingin a companion animal, comprising:

-   (a) a dietary composition; and-   (b) a feeding manual providing written instructions for an owner of    said animal.

Specifically, the feeding manual may include information on: (1)optionally, method of assessing severity of the idiopathic vomitingcondition of the companion animal; (2) frequency of feeding; (3)duration of feeding; (4) method of feeding; and (5) optionally, methodof monitoring the idiopathic vomiting condition of the companion animalto determine when to stop the feeding.

These and other aspects of the present invention will become moreapparent upon reading the following detailed description and examples ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 39 show the 5-HT_(3a) dosage response curves of various plantmaterials and extracts.

FIGS. 40 to 44 are graphs showing the unexpected synergistic 5-HT_(3a)inhibitory effect achieved by certain combinations of plant materials orextracts.

FIGS. 45 to 64 show the NK-1 dosage response curves of various plantmaterials and extracts.

FIG. 65 is a graph showing the unexpected synergistic NK-1 inhibitoryeffect achieved by a specific combination of plant materials orextracts.

DETAILED DESCRIPTION

Except as otherwise noted, the articles “a”, “an”, and “the” mean “oneor more.” The term “comprising” means that other steps and otheringredients which do not affect the end result can be added, and thisterm encompasses the terms “consisting of” and “consisting essentiallyof” The compositions and methods/processes of the present invention cancomprise, consist of, and consist essentially of the essential elementsand limitations of the invention described herein, as well as any of theadditional or optional ingredients, components, steps, or limitationsdescribed herein. All percentages, parts and ratios are based upon thetotal weight of the compositions of the present invention, unlessotherwise specified. All such weights as they pertain to listedingredients are based on the active level and, therefore do not includecarriers or by-products that may be included in commercially availablematerials. All ratios are weight ratios unless specifically statedotherwise. All temperatures are in Celsius degrees, unless specificallystated otherwise. All dimensions and values disclosed herein (e.g.,quantities, percentages, portions, and proportions) are not to beunderstood as being strictly limited to the exact numerical valuesrecited. Instead, unless otherwise specified, each such dimension orvalue is intended to mean both the recited value and a functionallyequivalent range surrounding that value. For example, a dimensiondisclosed as “40 mm” is intended to mean “about 40 mm.”

Herein, the term “treat,” “treating” or “treatment” covers all mannersof treatment of a disease or condition in the animal of interest, whichincludes: (i) inhibiting the disease or condition, i.e., completelyarresting its development; (ii) reducing the disease or condition, i.e.,causing regression of the disease or condition; and (iii) alleviating orrelieving the symptoms resulting from the disease or condition, i.e.,relieving pain or suffering without addressing the underlying disease orcondition.

The term “effective” means an amount of a subject active high enough toprovide a significantly positive modification of the condition to betreated. An effective amount of the subject active will vary with theparticular condition being treated, the severity of the condition, theduration of the treatment, the nature of concurrent treatment, and likefactors.

The term “vomit” or “vomiting” as used herein cover any voluntary orinvoluntary expulsion of contents from either one's stomach or esophagusinto the mouth and sometimes into the nose. Such acts can be referred tovariously as vomiting, regurgitation, emesis, throwing up, puking,heaving, and the like, but are collectively covered herein by the sameterm “vomit” or “vomiting.”

The term “feline companion animal” or “feline” as used herein broadlycovers all animals in the Felidea family that can potentially be takenin by humans as either indoor or outdoor companions, which include, butare not limited to: domestic cats, cougars, cheetahs, lynxes, ocelots,tigers, lions, jaguars, panthers, leopards, and the like. The term“companion animal” as used herein includes, but is not limited to:feline companion animals as described hereinabove; all animals in theCanidea family that can potentially be taken in by humans as eitherindoor or outdoor companions, such as domesticated dogs (Canisfamiliaris), wolves, foxes, jackals, coyotes, and the like; othersmaller domestic mammals, such as ferrets, raccoons, rabbits, mice,rats, hamsters, guinea pigs, and the like.

The present invention has identified a specific group of plant materialsor extracts thereof that are particularly effective as inhibitors of5-HT_(3a) and/or NK-1 receptor in felines and canines. Such plantmaterials or extracts are naturally derived with little or no sideeffects on the companion animals. Further, such plant materials orextracts have improved tastes or smells in comparison with syntheticcompounds. Therefore, these plant materials or extracts can be readilyused to formulate dietary compositions or regiments for managing andtreating idiopathic vomiting in canines and felines. These plantmaterials or extracts may also be used in managing or treatingidiopathic vomiting in other companion animals, or in other mammals,including in humans.

As used herein, the term “effective as inhibitors of 5-HT_(3a) and/orNK-1 receptor” means a5-HT_(3a) and/or NK-1 receptor affinity (IC₅₀) ofless than 100 ppm, preferably less than 50 ppm, and more preferably lessthan 10 ppm. To determine the 5-HT_(3a) and/or NK-1 receptor affinity,various receptor binding assays well known in the art may readily beused.

Botanicals with 5HT_(3a) Receptor Inhibitory Effect

-   -   It has been discovered by the inventors of the present invention        that the following plant materials or extracts thereof have        surprisingly high 5-HT_(3a) receptor inhibitory effect with        little or no cell toxicity:    -   Leptospermum Scoparium (lemon tea tree) leaf extract;    -   a water-soluble fraction of Melaleuca alternifolia (tea tree)        oil;    -   Rheum palmatum (rhubarb);    -   Lavandula angustifolia (lavender tea tree) extract;    -   Fusanus Spicatus (Australian sandalwood) extract;    -   Santalum Album (sandalwood) oil;    -   Evodia Rutaecarpa (evodia) fruit;    -   Coptis chinensis (coptis) rhizome;    -   oils from Wisteria sinensis;    -   Myrtus Communis (myrtle) extract;    -   Prunus serotina (wild cherry) bark;    -   Aquilaria sinensis (Chinese Agarwood) resinous heartwood        extract;    -   Zingiber officinale (ginger) root;    -   Calendula officinalis (marigold);    -   Boswellia serrata (boswellia);    -   oil from Boswellia carterii (frankincense oil);    -   Zanthoxylum americanum (prickly ash fruit) extract;    -   Pinus pinaster bark extract;    -   Marrubium vulgare (horehound);    -   Citrus aurantium (bitter orange) fruit;    -   Angelica archangelica (angelica);    -   Cocos nucifera (coconut) oil;    -   Rosa chinensis (Chinese rose);    -   Cinnamomum aromaticum (cinnamon);    -   Vaccinium myrtillus (bilberry) extract;    -   Vaccinium corymbosum (blueberry) leaf extract;    -   Daucus carota (carrot) extract;    -   Anthemis eecutita (chamomile) extract;    -   Curcuma longa (turmeric) extract;    -   Vaccinium macrocarpon (cranberry) extract;    -   Linum usitatissimum (flax) oil powder;    -   Hovenia dulcis (Japanese raisin tree) seed extract;    -   Andrographis paniculata (Andrographis herb) extract;    -   Bee pollen extract;    -   Eucalyptus radiata (Australia eucalyptus) extract;    -   Yerba santa extract;    -   Piper nigrum (black pepper) oil;    -   Ginkgo biloba extract; and    -   Capsicum annuum (cayenne) extract.

Further, it has been discovered that the following combinations of plantmaterials or extracts act in synergy as inhibitors of 5-HT_(3a)receptor:

-   -   Curcuma longa (turmeric) and Glycyrrhiza glabra (licorice);    -   Turmeric, licorice, and ginger;    -   Turmeric and ginger;    -   Turmeric and rhubarb; and    -   Licorice, ginger, and rhubarb.        Botanicals with NK-1 Receptor Inhibitory Effect

It has been discovered by the inventors of the present invention thatthe following plant materials or extracts thereof have surprisingly highNK-1 receptor inhibitory effect with little or no cell toxicity:

-   -   Podophyllum peltatum (podophyllin);    -   Rhodiola rosea (rhodiola);    -   Tanacetum parthenium (feverfew);    -   Pinus massoniana (pink bark);    -   Boswellia serrata (boswellia);    -   Dracaena cinnabari (Dragon's Blood) extract;    -   Tilia vulgaris (linden);    -   Paullinia cupana (guarana);    -   Uncaria tomemtosa (cat's claw);    -   Hypericum perforatum (St. John's wort);    -   Calendula officinalis (calendula);    -   Camellia sinensis (green tea) extract;    -   Vitis vinifera (grape) extract;    -   Cinnamomum aromaticum (cinnamon) extract;    -   Curcuma longa (turmeric) extract;    -   Fructus forsythiae (forysthia fruit) extract;    -   Glycyrrhiza glabra (licorice) extract;    -   Litsea cubeba oil;    -   Melaleuca alternifolia (tea tree) oil; and    -   Phyllanthus emblica (amla) extract.

Further, it has been discovered that the combination of turmeric andlicorice acts in synergy as inhibitors of NK-1 receptor.

Methods of Administration

The above-listed plant materials or extracts can be administered by anywell-known delivery method, which includes, but is not limited to: oraldelivery, inhalation, rectal injection, or parenteral delivery, such as,for example, topical application, transdermal application, intravenousinjection, subcutaneous injection, intra-muscular injection, and thelike. Such plant materials or extracts can be administered alone or incombination with any acceptable carriers or diluents to formcompositions such as dry kibbles, wet canned food, gravies, treats,tablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like.

In a preferred but not necessary embodiment of the present invention,such plant materials or extracts are orally administered to a companionanimal, for example, as a part of a dietary composition for thecompanion animal. Such a dietary composition can be a liquid, a solid,or a semi-solid. Further, the dietary composition may be formulated aseither a pet food that is fed to the animal at meal times, or a pet foodsupplement that is fed to the animal either separately from or incombination with the pet food for the animal.

As a pet food, the dietary composition may comprise a nutritionallycomplete diet for the intended recipient companion animal. Anutritionally complete diet contains known required nutrients to sustainlife of the companion animal in proper amounts and proportions based onrecommendations of well recognized authorities, including governmentalagencies such as United States Food and Drug Administration's Center forVeterinarian Medicine, the American Feed Control Officials Incorporated,with the exception of water.

For example, a pet food composition of the present invention maycomprise at least a source of carbohydrate, a source of protein, andoptionally a source of fat. More preferably, the pet food compositionprovides the companion animal with a nutritionally complete and balanceddiet, which may comprise: from about 1% to about 99%, preferably fromabout 1% to about 90% and more preferably from about 5% to about 45%, byweight of carbohydrates; from about 5% to about 99.9%, preferably fromabout 10% to about 90% and more preferably from about 20% to about 60%,by weight of protein; from about 0.1% to about 50%, preferably fromabout 1% to about 40% and more preferably from about 5% to about 20%, byweight of fat; from about 0.01% to about 20%, preferably from about 1%to about 11%, by weight of dietary fiber; from about 0.01% to about 15%,preferably from about 0.1% to about 10% and more preferably from 1% to8%, by weight of vitamins, minerals, antioxidants, and other nutrientssupporting the needs of the companion animal. The carbohydrates can beprovided by grains such as rice, corn, milo, sorghum, barley, wheat,oats and the like. The protein can be provided by either animal-derivedsources, such as meats (beef, pork, lamb, poultry, fish, and the like),eggs, and milk, or plant-derived sources, such as soybean, cereals,cottonseed, peanut, and the like. The dietary fibers can be provided bycellulose, hemicellulose, pectin, lignin, and gums. Further, the petfood composition of the present invention may contain variousingredients typically used in pet foods, such as fillers, flavors,binding agents, thickeners, stabilizers, emulsifiers, sweeteners,food-grade colorants, buffers, salts, and the like. Particularlypreferred binding agents and/or thickeners are gelatin, celluloseethers, starch, starch esters, starch ethers, and modified starches.

The pet food composition can be formulated as dry kibbles, wet cannedfoods, gravies, or treats. It may be fed to the companion animal on adaily basis, either at regular meal times (such as, for example, fromonce a time up to six times a day) or continuously throughout the day asneeded (for example, through an automatic feeder or by simply providingan excessive amount). The composition may be fed ad libitum.

In certain embodiments, the pet food composition is a treat. Treats asused herein refer to pet food compositions that are given to thecompanion animal to entice the animal to eat during a non-meal time.Treats may also have nutritional value and have a food-like compositionincluding one or more nutrients as described hereinabove, but are not inthemselves a nutritionally complete diet.

The dietary composition of the present invention can also be provided asa pet food supplement, which is used with another feed, eitherconcurrently or separately, to improve the nutritive balance orperformance of the companion animal. Pet food supplements include, butare not limited to, any composition that is fed undiluted in addition toother feeds, thereby offering free choice with other parts of ananimal's ration that are separately available, or is diluted and mixedwith an animal's regular feed to produce a complete feed. TheAssociation of American Feed Control Officials (AAFCO) providesguidelines, for example, that contain a discussion relating tosupplements, which can be in various forms including powders, liquids,syrups, pills, encapsulated compositions, and the like. Further, the petfood supplement can be provided as a part of a toy for the companionanimal, with partially or fully consumable components.

The dietary compositions as described above can be readily formed bymixing one or more above-listed plant materials or extracts capable ofinhibiting the 5-HT_(3a) and/or NK-1 receptor with one or moreabove-disclosed dietary nutrients suitable for a companion animal.

To determine an efficacious dosage, multiple complete cross-over studiescan be performed with the plant materials or extracts in the companionanimal to be treated at various doses. The optimal dose is selectedbased on the maximal ability to reduce or eliminate idiopathic vomitingin companion animals exhibiting perceivable symptoms of idiopathicvomiting. When administered to the companion animal in form of a petfood composition, the plant materials or extracts as describedhereinabove are preferably administered in dosages ranging from 0.1 ppmto 75,000 ppm, preferably from 1 ppm to 1000 ppm. When administered inform of a supplement, the plant materials or extracts as describedhereinabove are preferably administered in dosages ranging from 0.1% to99%, preferably from 0.5% to 15%, by weight of the supplement. Thecomposition is preferably a dietary composition, but can be any othercomposition, which includes, but is not limited to: topicalcompositions, injectable compositions, nasal compositions, rectalcompositions, and the like.

Frequency and duration of the administration can be varied depending onthe animal's condition, the species of animal being treated, itsindividual response to the treatment, and the type of pharmaceuticalformulation chosen. Frequency can range from once a month to six times aday, preferably from once a week to four times a day, and morepreferably from once a day to three times a day. Duration can range fromfive days to the entire life span of the animal, e.g., twenty fiveyears. Preferably, the duration ranges from one week to fifteen years,more preferably from two weeks to one year, and most preferably from onemonth to six months.

Other kinds of vomiting or regurgitation may occur concurrently withidiopathic vomiting. Compositions of the present invention may includefeatures to reduce other causes of vomiting. For example, a foodcomposition may include large food particles relative to the size of thesubject animal's mouth, to discourage rapid eating that may causeregurgitation; or may include ingredients to reduce the occurrence ofhairballs, such as proteases, polyol fatty acid polyesters, laxatives,and the like; or may include ingredients to promote gastrointestinalhealth, such as prebiotics or probiotics. Compositions of the presentinvention may include medicinal drugs with anti-emetic activity.

The present invention also covers an article of commerce, preferably inform of a kit, containing the dietary composition as describedhereinabove together with instructions that provide information on howto orally administer or feed the dietary composition to the companionanimal. Specifically, the instructions may provide information on, forexample: assessing the severity of the idiopathic vomiting condition ofthe companion animal; frequency of feeding; duration of feeding; mode offeeding; and monitoring the idiopathic vomiting condition of thecompanion animal to determine when to modify the frequency and/or theduration of feeding.

Any standard packaging that is suitable for delivery and sale of thedietary compositions as disclosed herein can be used in forming the kit.The kit can also include specific written benefit statements related tothe prevention, reduction, and elimination of idiopathic vomiting oremesis in companion animals. The benefit statements can also relate tothe health benefits resulted from such prevention or treatment ofidiopathic vomiting or emesis, such as increased body weight and energylevel, improved immune functions, and prolonged life span.

The present invention is illustrated by the following examples. It willbe understood, however, that the invention is not limited to thespecific details of these examples.

EXAMPLES

Assays for Screening 5-HT_(3a) Receptor Inhibitors

Various known high through-put screening assays can be used to determinethe inhibitory effects of a material on 5-HT_(3a) receptors.

For example, the 5-HT_(3a) receptor is a ligand-gated, non-selectivecation channel located in the central and peripheral nervous system.Activation of the 5-HT_(3a) receptor followed by rapid depolarization ofthe peripheral or central neuron causes a rapid rise in cytosolic Ca²+and Na⁺ concentration by inducing calcium and sodium influx andmobilization of intracellular calcium stores, as well as modulating therelease of various neurotransmitters and neuropeptides such as dopamine,cholecystokinin, acetylcholine, GABA, substance P or serotonin itself.Due to activation of 5-HT_(3a) receptor following calcium and sodiuminflux and the subsequent rapid intracellular calcium and sodiumincrease, a calcium indicator (e.g., Fluo-4 AM) or a sodium dye cantherefore be readily used to detect influx Ca⁺² or Na⁺ signal using theFluorometric Imaging Plate Reader (FLIPR) assay to identify agonists orantagonists of 5-HT_(3a) receptors.

The 5-HT_(3a) FLIPR assay can specifically be conducted by the followingsteps. First, HEK-23 (human embryonic kidney) cells stably expressedwith h5HT3A receptor are grown in 16-18 ml growth medium in a 75 cm²flask for 2-3 days at 37° C. in a mammalian cell culture incubator with5% CO₂ and 90% humidity. The growth medium may contain, for example,DMEM/F12 (1:1, Invitrogen 11039) supplemented with 10% FBS (fetal bovineserum), 100 μg/ml Antibiotic/Antimycotic, and 150 ug/ml G418. The cellmedium is then transferred to a 50 ml tube, and the cells are washedwith 10 ml PBS. Subsequently, 2 ml of 0.05% Trypsin-EDTA is added todetach cells, and the above cell medium is added back to flask toinactivate trypsin. Next, the cells are transferred back to the above 50ml tube, which is centrifuged at 850 rpm for 3 minutes to remove medium.The cells obtained from centrifugation are then re-suspended with growthmedium at 1-1.5 ml per flask cells. One vial of Fluo-4 AM (calciumindicator, 50 ug) is subsequently dissolved with 20 ul of PluronicF-127, and 10 ul of Fluo-4 AM solution per flask cells is added (1 vialof Fluo-4 AM solution, 20 ul, is good for 2 flask of cells). The cellsare then stained with Fluo-4 AM for 30 min at room temperature withgently shaking on a shaker, followed by addition of 45 ml of the assaybuffer [HBSS with CaCl₂ and MgCl₂ (Invitrogen 14025), 20 mM HEPES, pH7.2] to wash cells once. Centrifugation is carried out once more at 850rpm for 3 minutes to remove assay buffer. The resulting cell pellet isagain re-suspended in Assay buffer (per flask cells with 18-20 ml assaybuffer). Ninety microliters of cells (50K cells/well) is loaded in the96-well plates that are pre-loaded with the plant materials or extractsto be tested (10 ul of 1 mM test materials, the final concentration ofthe test materials will be 100 uM). The plates are placed at roomtemperature for 15-30 min in dark and then transferred to FLIPR-384instrument (Molecular Devices). The master plate containing 6× ofagonist (60 uM serotonin) is placed, and all test plates are read afteradding agonist. The calcium signal of the test plates is finallyrecorded by the FLIPR program. The average and standard deviations arecalculated using Excel, and the background (buffer) is subtracted. Thepercentage (%) inhibition is then calculated as

$\left( {1 - \frac{{Test}\mspace{14mu}{Material}}{{Agonist}\mspace{14mu}{Control}}} \right) \times 100.$A test material will be considered as having an inhibitory effectagainst the 5-HT_(3a) receptor if the percentage inhibition calculatedis greater than 40%.

Alternatively, a cell-based serotonin receptor assay can be used toscreen inhibitors of the 5-HT_(3a) receptor. Using the agonist serotoninand cells co-transfected to over express the 5-HT_(3a) receptor and theluminescent aequorin calcium sensitive reporter, this assay can beconducted to identify suitable new actives targeting the 5-HT_(3a)receptor with great sensitivity, scalability and specificity.

Aequorin is a photo-protein originating from the Jellyfish AequoreaVictoria. It is initially translated as an apo-enzyme requiring thehydrophobic group coelenterazine to initiate the conversion to aequorin.Upon binding of calcium, the coelenterazine is oxidized by aequorin intocoelenteramide (BFP) resulting in the emission of blue light and CO₂.Therefore, it is particularly useful for visualizing or detecting influxCa⁺² signal.

The aequorin-assisted serotonin receptor assay can be carried out withthe following steps. Cyro-preserved Human Cells (HEK293 parent line),co-expressing Serotonin 5HT_(3a) receptor and the Aequorin calciumsensor, γ-Irradiated (Perkin Elmer, Cat. No. ES-402-AF) are thawed andcultured 18-24 hours in DMEM/F12 with Hepes buffer, no phenol red+10%FBS without antibiotics (Invitrogen, Cat. No. 11039-021). After 18-24 hof culture, the cells are detached gently by flushing with their cellculture medium. Washing with an additional 10 mLs of cell culture mediawill ensure that optimum number of cells is captured. The cells are thencentrifuged at 150×g, counted and re-suspended at 1×10{circumflex over( )}6 cells/mL in BSA medium [DMEM/Ham's F12 (with 15 mM HEPES,L-glutamine, without phenol red) culture medium (Invitrogen, Cat. No.11039-021)+10% protease-free BSA (Sigma Aldrich, Cat. No. A9205) in H₂Owith a final BSA concentration of 0.1%] in a Falcon tube. Coelenterazineis added at a final concentration of 5 μM in assay medium. Ascoelenterazine stock solution is in methanol, it is mixed well whileadding the coelenterazine solution to the cell suspension to avoiddamaging the cells. The 10 mL Falcon tube is then wrapped in aluminumfoil and placed on a rotating wheel (about 45° angle and 7 rpm). Thecells are subsequently incubated from 4 hrs to 18 hrs at ˜20° C.(temperature should remain below 25° C.). On the day of the assay, cellsare diluted in BSA medium to a final concentration of 2.0×10{circumflexover ( )}5 cells/mL. The cells are incubated again for at least 1 h atroom temperature. The screen plates are then prepared. Antagonists arediluted in BSA medium referenced above at 2× concentration, and 50 μlare dispensed per well.

Fifty microliters of cell suspension (2.0×10{circumflex over ( )}5/mlfor a final concentration of 1.0×10{circumflex over ( )}4/well) is addedinto the antagonist wells and then incubated for 15 minutes in the darkat room temperature. Only one plate is prepared at time. Fiftymicroliters of serotonin (3×EC80 (EC stands for Effective Concentration)concentration (30 μM) to get 1×EC80 (10 μM) final concentration) isinjected using the plate readers injectors into the mix of cells andantagonist, and the light emitted is recorded for 10 s. An 8 point dosecurve was conducted using the agonist serotonin to determine the EC50for this system. Doses ranged from 3.9E-7 to 5E-5 [M]. The resultingEC50 was determined to be 1.925E-6±3.715E-6 [M].

FIGS. 1 to 39 show the 5-HT_(3a) dosage response curves of plantmaterials and extracts described hereinabove with high 5-HT_(3a)inhibitory effect. Specifically, FIG. 1 shows the 5-HT_(3a) dosageresponse curve of lemon tea tree leaf extract. FIG. 2 shows the5-HT_(3a) dosage response curve of a water-soluble fraction of tea treeoil. FIG. 3 shows the 5-HT_(3a) dosage response curve of rhubarb. FIG. 4shows the 5-HT_(3a) dosage response curve of lavender tea tree extract.FIG. 5 shows the 5-HT_(3a) dosage response curve of Australiansandalwood extract. FIG. 6 shows the 5-HT_(3a) dosage response curve ofsandalwood oil. FIG. 7 shows the 5-HT_(3a) dosage response curve ofevodia fruit. FIG. 8 shows the 5-HT_(3a) dosage response curve of coptisrhizome. FIG. 9 shows the 5-HT_(3a) dosage response curve of oils fromWisteria sinensis. FIG. 10 shows the 5-HT_(3a) dosage response curve ofmyrtle extract. FIG. 11 shows the 5-HT_(3a) dosage response curve ofwild cherry bark. FIG. 12 shows the 5-HT_(3a) dosage response curve ofChinese Agarwood resinous heartwood extract. FIG. 13 shows the 5-HT_(3a)dosage response curve of ginger root. FIG. 14 shows the 5-HT_(3a) dosageresponse curve of marigold. FIG. 15 shows the 5-HT_(3a) dosage responsecurve of boswellia. FIG. 16 shows the 5-HT_(3a) dosage response curve offrankincense oil. FIG. 17 shows the 5-HT_(3a) dosage response curve ofprickly ash fruit. FIG. 18 shows the 5-HT_(3a) dosage response curve ofPinus pinaster bark extract. FIG. 19 shows the 5-HT_(3a) dosage responsecurve of horehound. FIG. 20 shows the 5-HT_(3a) dosage response curve ofbitter orange fruit. FIG. 21 shows the 5-HT_(3a) dosage response curveof angelica. FIG. 22 shows the 5-HT_(3a) dosage response curve ofcoconut oil. FIG. 23 shows the 5-HT_(3a) dosage response curve ofChinese rose. FIG. 24 shows the 5-HT_(3a) dosage response curve ofcinnamon. FIG. 25 shows the 5-HT_(3a) dosage response curve of bilberry.FIG. 26 shows the 5-HT_(3a) dosage response curve of blueberry leaf.FIG. 27 shows the 5-HT_(3a) dosage response curve of carrot extract.FIG. 28 shows the 5-HT_(3a) dosage response curve of chamomile. FIG. 29shows the 5-HT_(3a) dosage response curve of turmeric. FIG. 30 shows the5-HT_(3a) dosage response curve of cranberry. FIG. 31 shows the5-HT_(3a) dosage response curve of flax oil powder. FIG. 32 shows the5-HT_(3a) dosage response curve of Japanese Raisin tree seed extract.FIG. 33 shows the 5-HT_(3a) dosage response curve of Andrographis herb.FIG. 34 shows the 5-HT_(3a) dosage response curve of bee pollen. FIG. 35shows the 5-HT_(3a) dosage response curve of Australian eucalyptusextract. FIG. 36 shows the 5-HT_(3a) dosage response curve of Yerbasanta. FIG. 37 shows the 5-HT_(3a) dosage response curve of black pepperoil. FIG. 38 shows the 5-HT_(3a) dosage response curve of Ginkgo biloba.FIG. 39 shows the 5-HT_(3a) dosage response curve of cayenne.

Further, FIGS. 40-44 are graphs showing the synergistic 5-HT_(3a)inhibitory effect achieved by certain combinations of plant materials orextracts as described hereinabove. Specifically, FIG. 40 shows thecombined 5-HT_(3a) inhibitory effect achieved by the combination ofturmeric (T) and licorice (L), which is larger than the sum of the5-HT_(3a) inhibitory effects achieved separately by turmeric andlicorice. FIG. 41 shows the combined 5-HT_(3a) inhibitory effectachieved by the combination of turmeric (T), licorice (L) and ginger(G), which is larger than the sum of the 5-HT_(3a) inhibitory effectsachieved separately by turmeric, licorice and ginger. FIG. 42 shows thecombined 5-HT_(3a) inhibitory effect achieved by the combination ofturmeric (T) and ginger (G), which is larger than the sum of the5-HT_(3a) inhibitory effects achieved separately by turmeric and ginger.FIG. 43 shows the combined 5-HT_(3a) inhibitory effect achieved by thecombination of turmeric (T) and rhubarb (R), which is larger than thesum of the 5-HT_(3a) inhibitory effects achieved separately by turmericand rhubarb. FIG. 44 shows the combined 5-HT_(3a) inhibitory effectachieved by the combination of licorice (L), ginger (G) and rhubarb (R),which is larger than the sum of the 5-HT_(3a) inhibitory effectsachieved separately by licorice, ginger and rhubarb.

Assays for Screening NK-1 Receptor Inhibitors

Various known high through-put screening assays can also be used todetermine the inhibitory effects of a material on the NK-1 receptors.

One exemplary assay is the Tango™ G-Protein Coupled Receptors (GPCR)cell-based assays by Invitrogen. First, TACR1-bla U2OS cells (fromInvitrogen) are cultured in McCoy's medium. The cultured cells are thenplated in DMEM into 96-well plates (15,125 cells/well in 90 uL/well).After 16-24 hours, the cells are treated with test materials or positivecontrol antagonist (which is 100 nM Aprepitant) and then incubated for30-60 minutes at 37° C. in 5% CO2. Subsequently, the cells are treatedwith 1 nM SAR9 Substance P agonist and incubated again for 5 hours at37° C. in 5% CO2. The plates are removed from the incubator andequilibrated at room temperature for 15 minutes. During theequilibration step, the LiveBlazer substrate detection solution (fromInvitrogen) is prepared. Six times of substrate mixture is added to eachwell, followed by incubating the plates in the dark at room temp for 2hours. The plates are read by the Envision microplate reader in 2channels using the “Geneblazer 451” protocol, which include a bluechannel (Excitement at 405 and Emission at 460) and a green channel(Excitement at 405 and Emission at 535).

FIGS. 45 to 64 show the NK-1 dosage response curves of plant materialsand extracts described hereinabove with high NK-1 inhibitory effect.Specifically, FIG. 45 shows the NK-1 dosage response curve ofpodophyllin. FIG. 46 shows the NK-1 dosage response curve of rhodiola.FIG. 47 shows the NK-1 dosage response curve of feverfew. FIG. 48 showsthe NK-1 dosage response curve of pink bark. FIG. 49 shows the NK-1dosage response curve of boswellia. FIG. 50 shows the NK-1 dosageresponse curve of dragon's blood. FIG. 51 shows the NK-1 dosage responsecurve of linden. FIG. 52 shows the NK-1 dosage response curve ofguarana. FIG. 53 shows the NK-1 dosage response curve of cat's claw.FIG. 54 shows the NK-1 dosage response curve of St. John's wort. FIG. 55shows the NK-1 dosage response curve of calendula. FIG. 56 shows theNK-1 dosage response curve of green tea. FIG. 57 shows the NK-1 dosageresponse curve of grape extract. FIG. 58 shows the NK-1 dosage responsecurve of cinnamon. FIG. 59 shows the NK-1 dosage response curve ofturmeric. FIG. 60 shows the NK-1 dosage response curve of forsythiafruit. FIG. 61 shows the NK-1 dosage response curve of licorice. FIG. 62shows the NK-1 dosage response curve of Litsea cubeba oil. FIG. 63 showsthe NK-1 dosage response curve of tea tree oil. FIG. 64 shows the NK-1dosage response curve of amla.

Further, FIG. 65 shows the synergistic NK-1 inhibitory effect achievedby certain combinations of plant materials or extracts as describedhereinabove. Specifically, FIG. 65 shows the combined NK-1 inhibitoryeffect achieved by the combination of turmeric (T) and licorice (L),which is larger than the sum of the NK-1 inhibitory effects achievedseparately by turmeric and licorice.

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A pet dietary composition comprising one or more plant materials or extracts thereof, wherein the plant materials or extracts thereof consist of Curcuma longa and one or more of licorice, ginger or rhubarb in amounts effective to result in a greater 5-HT3a and/or NK-1 receptor inhibition than the sum of the 5HT3a or NK-1 inhibitory effects achieved by each plant material or extract, separately.
 2. The pet dietary composition of claim 1, wherein the composition is a pet food.
 3. The pet dietary composition of claim 2, wherein the pet food is in the form of dry kibbles, wet canned food, gravies, or treats.
 4. The pet dietary composition of claim 1, wherein the composition is a pet supplement comprising from 0.1% to 99% of said one or more plant materials or extracts thereof by weight.
 5. The pet dietary composition of claim 4, wherein the pet supplement comprises from 0.5% to 15% of said one or more plant materials or extracts thereof by weight.
 6. The pet dietary composition of claim 4 wherein the pet supplement is in the form of a powder, liquid, syrup, pill, encapsulated composition, or combinations of these.
 7. The pet dietary composition of claim 1, wherein the plant materials or extracts thereof consist of Curcuma longa and licorice, and one or more of ginger or rhubarb.
 8. The pet dietary composition of claim 1, wherein each plant material or extract is present in an amount of at least 100 μM. 